Evaluation of Microorganisms Cultured from Injured and Repressed Tissue Regeneration Sites in Endangered Giant Aquatic Ozark Hellbender Salamanders

Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969–2009, and the abnormality/injury rate and apparent lack of regeneration were established. Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a Cryptobranchid salamander. The incidence of abnormalities/injury and retarded regeneration in C. a. bishopi may have many contributing factors including disease and habitat degradation. Results from this study may provide insight into other amphibian population declines

Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the Rag GTPases

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain–containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1’s GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex

Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex - comprising DEPDC5, NPRL2 and NPRL3 - regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.National Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (R01 CA129105)National Institutes of Health (U.S.) (R37 AI047389)United States. Department of Defense (W81XWH-15-1-0230)National Science Foundation (U.S.) (fellowship 2016197106

Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex - comprising DEPDC5, NPRL2 and NPRL3 - regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.National Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (R01 CA129105)National Institutes of Health (U.S.) (R37 AI047389)United States. Department of Defense (W81XWH-15-1-0230)National Science Foundation (U.S.) (fellowship 2016197106